The Region X Northwest Regional Center of Excellence (NWRCE) for Biodefense and Emerging Infectious Diseases is an essential component in a nationwide network of biomedical research programs to combat infectious disease threats to the population. The NWRCE is currently a highly interactive and thematic program focused on Gram-negative bacterial pathogens. The same general consistent focus has existed since its inception. There are three themes: (1) mechanisms of Gram-negative bacterial pathogenesis, (2) innate immune responses to Gram-negative bacterial pathogens, and (3) translation of (1) and (2) into early stage therapeutic development to prevent or treat Gram-negative bacterial diseases. The NWRCE is centered at the University of Washington (UW) in Seattle;in this renewal application, multiple sites are proposed in Seattle, two in Oregon, one in Idaho, and one in Maryland. The NWRCE has included the NIAID Rocky Mountain Laboratory since its inception and this relationship will continue and grow as part of this renewal as collaborations are expanded to include tularemia as well as plague research. British Columbia is also in our geographic region and collaboration is proposed with the University of Victoria, British Columbia. The NWRCE renewal proposes three international sites: Thailand, Sweden, and Mexico. The projects, cores, domestic and international sites, as well as collaborators, are highly interactive. The individuals that make up this proposal have worked together for many years during the last funding period and before the RCE was funded. The majority of investigators newly added to this to this application for renewed funding of the NWRCE have had long collaborative relationships with members of the center. The interactive nature and focus of the NWRCE is a major strength of the program and allows the NWRCE to rapidly and nimbly respond to national priorities to achieve important objectives with respect to Gram-negative bacterial infections. The Seattle biomedical community occupies a unique international leadership position in infectious diseases research and global health and hence centering such a program at the UW is logical for both national and regional needs. The UW Medical School has traditionally emphasized its infectious disease research program. The training programs in infectious diseases, microbiology, immunology, global health, and genome sciences are highly successful, interactive, and comprehensive. The UW currently has five NIH funded training grants in infectious diseases research that have trained hundreds of individuals over the last 30 years, many of them now leaders in the field. Infectious diseases research at the UW permeates many of the clinical and basic science departments, including Global Health, Medicine, Pediatrics, Genome Sciences, Microbiology, Immunology, Biochemistry, and Structural Biology. Currently, more than four hundred trainees are engaged in infectious disease research in these departments. Furthermore, these departments host a variety of centers and cooperative research programs studying infectious diseases, such the Center for AIDS Research and the Sexually Transmitted Diseases Cooperative Research Center, and the Department of Global Health, all directed by Dr. King Holmes. Other centers include the AIDS Vaccine Evaluation Unit directed by Dr. Julie McElrath, the Cooperative Research Center for Structural Genomics of Pathogenic Protozoa directed by Dr. Wim Hoi, and the Cystic Fibrosis Research and Development Program directed by Dr. Peter Greenberg. These centers involve cooperative research consortia that have been assembled from a variety of departments to study specific infectious disease problems. The cooperative research culture and environment at the UW greatly facilitated development of the NWRCE, since a cooperative spirit and culture was required to establish the NWRCE as a thematic interactive center. Such a cooperative environment will be essential for continued success in achieving the programmatic goals of the NIAID and the national infectious diseases agenda. There are many institutions in the region with established connections to the UW that also have strong microbiology and infectious diseases research programs, including many that participate and collaborate with the NWRCE. These institutions include the Fred Hutchinspn Cancer Research Center, the Children's Hospital and Regional Medical Center, the Institute for Systems Biology, PATH, the Infectious Disease Research Institute, and the Seattle Biomedical Research Institute (containing the NIAID funded structural biology center which collaborates with the NWRCE) among others. Other important institutions with infectious disease research programs in the region include the Rocky Mountain Laboratories of the NIAID, Pacific Northwest National Laboratories (Containing an NIAID funded Proteomics Center), the Universities of Alaska, Idaho, and Oregon, the Oregon Health Sciences Center, Oregon State University, Montana State University, and Washington State University. The majority of these institutions have been funded in the last five years through the NWRCE. Investigators at Montana State were initially funded through the NWRCE and then became an established component of the Rocky Mountain RCE (Dr. David Pascual). Investigators at the Guillemen). The Pacific Northwest (and specifically Seattle) is also one of the centers in the United States for biotechnology companies, many of which focus on infectious diseases and inflammation research, including Amgen (formerly Immunex). The geography of the NWRCE region is also uniquely important for biodefense and emerging infectious diseases in that it is situated on the northwestern coast of the United States, bordering Canada and serving as a transportation portal to Asia. This unique group of NWRCE-associated institutions forms a powerful base of resources for the NWRCE. In addition, the NWRCE has established important international collaborations to facilitate its disease-based activities in characterization of pathogen diversity with the establishment of centers in Thailand, Sweden, and Mexico to collaborate with NWRCE investigators on projects in melioidosis, tularemia, and enteric and other emerging Gram-negative bacterial infectious diseases. As a central hub in this network of affiliations, and as a leader in infectious diseases research, the NWRCE is prepared to continue to advance its role as a world leader in Gram-negative bacterial pathogenesis, immune responses, and therapeutic development and to train the next generation of researchers in this important area. There is a strong rationale for a continued program primarily focused on Gram-negative agents. There are several reasons why Gram-negative bacteria should be an important focus of study. Gram-negative bacteria have a common surface structure with two membranes separated by a periplasmic space. Two of the NIAID-classified Category A agents on the biodefense agenda are Gram-negative bacteria, as are the majority of Category B agents. Human infection with Gram-negative bacteria can be readily acquired through inhalation and oral ingestion, both routes that can be easily utilized in a biological attack. Gram-negative bacterial antibiotic resistance is increasing and, in contrast to Gram-positive bacteria, pharmaceutical companies have little in the drug development pipeline for these organisms. Many Gram-negative bacteria are also pathogens of food animals and could be used to attack food sources. Additionally, as important plant pathogens, Gramnegative bacteria also represent a significant threat to food crops, either as naturally emerging pathogens or as agents of biological terrorism. A variety of enteric and respiratory diseases have been utilized for many years as biological weapons because of their stability, potential for weaponization, and the ease with which they can be genetically engineered. In the book Biohazard, author Ken Alibek describes the former Soviet Union's biological weapons program, which focused on the Gram-negative organisms F. tularensis, Y. pestis, B. pseudomallei, B. mallei and Brucella abortus [1]. Alibek describes the development of two classes of genetically modified organisms: new antibiotic-resistant strains, and strains rendered more virulent by inclusion of toxin genes from another species. One example of the latter involved the addition of 8. pseudomallei toxin genes to F. tularensis. With the development of "super-bioweapons" using Gram-negative bacteria already an actuality, the need to expand our base of knowledge about the unique virulence factors and pathology of Gram-negative bacteria is particularly urgent. Consistent with this information, the Gram-negative bacteria Y. pestis (YP) and F. tularensis (FT) are main research foci of this proposal. Furthermore, because of its importance as an understudied pathogen that is endemic in Southeast Asia (to which the Northwest region serves as a transportation portal) and its possible use in biological attack, the Category B agent B. pseudomallei (BP) (and the closely related glanders agent B mallei, BM) will also be a research focus of the NWRCE. Research on Gram-negative bacteria such as [unreadable] cpli, Salmpnellae and Yersinia spp. has identified important conserved structural elements and mechanisms of virulence. The envelope of Gram-negative bacteria contains conserved molecules such as LPS, flagellin, and lipopeptides, which stimulate innate immune responses. The bacteria also contain conserved mechanisms to manipulate or modulate these responses by regulating the structure and synthesis of molecules such as LPS and flagellin and delivering enzymes which alter host responses. These organisms also have conserved mechanisms of resistance to killing by host innate immune effector molecules such as antimicrobial peptides and oxidative stress. Gramnegative bacteria have conserved mechanisms of secretion of virulence proteins that have conserved enzymatic activity to manipulate host processes and cause death. However, each pathogen has its unique complement of these properties and there is tremendous biological diversity among both the bacteria, which are constantly evolving, and humans, whose evolution was driven in part by bacterial pathogens and commensals. Therefore much more knowledge must be generated, particularly knowledge of current pathogens so that this knowledge can be effectively translated. This proposal is therefore focused on understanding the basic and unique biology of Gram-negative bacteria with a focus on these understudied organisms of national importance. Although YP, FT, and BP have some common features in being Gram-negative pathogens that infect the airway, they also have important differences. While all survive mammalian innate immune attack to promote disease, YP and FT have relatively non-inflammatory LPS structures [2, 3] while that of BP is inflammatory. YP inhibits phagocytosis [4] while FT and BP escape from the phagolysosome by lysis of the vacuole, where they activate cytosolic innate immune surveillance pathways [5]. Both YP and BP use multiple type III secretion systems as pathogenic strategies [6, 7], while FT uses a type II secretion system [8]. FT has a small genome [8] while BP has a large genome that is more closely related to Pseudomonas aeruginosa and Burkholderia 9-11]. FT and YP are transmitted by insect vectors [9, 11], and reside in small mammal reservoirs throughout the U.S. and the world. Interestingly, the attempt to reintroduce black ferrets into the wild regions of Montana in the summer of 2007 was limited by Y. pestis transmission from wild rodents which serve as a major ferret food source. BP has both plant and environmental reservoirs throughout Southeast Asia [10]. The nature and diversity of the organisms in these reservoirs must be understood for national security. It is also important to understand the nature of human diversity in response to these organisms so that in the case of national emergency priorities can be set and strategies can be developed that will utilize compounds which target our own immune systems to combat infectious diseases. While vaccines exist for FT and YP, these vaccines provide no protection against inhalation disease [12, 13]. The NWRCE developed early stage vaccine candidates in the last project period to combat these diseases. No vaccine exists for BP [14]. The NWRCE developed therapeutic candidates in the last project period that may have broad spectrum efficacy against these pathogens by augmenting innate immune responses and blocking bacterial secretion. The NWRCE will increase knowledge of the basic biology of these important infectious agents and translate this information into vaccines and compounds that will be broad spectrum therapeutics useful in the event of a biological attack and/or a naturally occurring outbreak. The unique qualifications of the NWRCE investigators in Gram-negative bacterial research and innate immune response provide an additional rationale for this proposal's focus.